David Cheerie, Margaret M. Meserve, Danique Beijer, Charu Kaiwar, Logan Newton, Ana Lisa Taylor Tavares, Aubrie Soucy Verran, Emma Sherrill, Stefanie Leonard, Stephan J. Sanders, Emily Blake, Nour Elkhateeb, Aastha Gandhi, Nicole S.Y. Liang, Jack T. Morgan, Anna Verwillow, Jan Verheijen, Andrew Giles, Sean Williams, Maya Chopra, Laura Croft, Hormos Salimi Dafsari, Alice E. Davidson, Jennifer Friedman, Anne Gregor, Bushra Haque, Rosan Lechner, Kylie-Ann Montgomery, Mina Ryten, Emil Schober, Gabriele Siegel, Patricia J. Sullivan, Ella F. Whittle, Bianca Zardetto, Timothy W. Yu, Matthis Synofzik, Annemieke Aartsma-Rus, Gregory Costain, Marlen C. Lauffer, the N=1 Collaborative
March 25, 2025
Summary
Of the around 7,000 known rare diseases worldwide, disease-modifying treatments are available for fewer than 5%, leaving millions of individuals without specialized therapeutic strategies. In recent years, antisense oligonucleotides (ASOs) have shown promise as individualized genetic interventions for rare genetic diseases. However, there is currently no consensus on which disease-causing DNA variants are suitable candidates for this type of genetic therapy. The patient identification working group of the N=1 Collaborative (N1C), alongside an international group of volunteer assessors, has developed and piloted consensus guidelines for assessing the eligibility of pathogenic DNA variants for ASO treatments. We herein present the N1C VARIANT (variant assessments toward eligibility for antisense oligonucleotide treatment) guidelines, including the guiding scientific principles and our approach to consensus building. Pathogenic, disease-causing variants can be assessed for the three currently best-established ASO treatment approaches: splice correction, exon skipping, and downregulation of RNA transcripts. A genetic variant is classified as “eligible,” “likely eligible,” “unlikely eligible,” or “not eligible” in relation to the different approaches or as “unable to assess.” We also review key considerations related to assessing the upregulation of transcripts from the wild-type allele, an emerging ASO therapeutic strategy. We provide additional tools and training materials to enable clinicians and researchers to use these guidelines for their eligibility assessments. With this initial edition of our N1C VARIANT guidelines, we provide the rare genetic disease community with guidance on how to identify suitable candidates for variant-specific ASO-based therapies and the possibility of integrating such assessments into routine clinical practice.
Introduction
There are around 7,000 different rare diseases known to date, with disease-modifying treatments approved for about 5% of them.1,2 A rare disease is defined as a condition that affects less than 200,000 people in the US or less than 1 in 2,000 individuals within Europe and Canada.1 It is estimated that 6% of the world’s population lives with a rare disease.3 The majority of rare diseases are thought to be genetic in origin, and with the massive improvements made in genetic diagnostics in the last decades, we can now diagnose up to 50% of individuals who suffer from a rare disease.4 As more individuals receive a molecular genetic diagnosis, the need to develop targeted treatments is increasingly urgent. However, because many of these rare diseases only affect a handful of individuals across the globe, the usual drug development route is not a viable pathway in most cases, and more bespoke therapeutic strategies are necessary.5
Antisense oligonucleotides (ASOs) are one promising form of genetic therapy. Over 20 different oligonucleotide therapies for general applications have been approved by the US Food and Drug Administration (FDA), the European Medicines Agency (EMA), the UK’s Medicines and Healthcare Products Regulatory Agency (MHRA), and/or the Japanese Ministry of Health, Labour and Welfare.6 Additionally, these drugs have been administered and well studied in thousands of people worldwide. Systemic delivery is possible (for instance via subcutaneous or intravenous injection), but localized or targeted delivery is also feasible for a growing number of target organs (brain and spinal cord via intrathecal injection, eye via intraocular injection, and liver and muscle via GalNAc and transferrin receptor targeting, respectively), allowing relatively low doses to be administered with potentially high treatment effects.7 Because of the finite half-life of ASOs, treatment needs to be administered repeatedly (often every 1–4 months), but this also allows the treatment regimen and dosing to be tailored to each individual where helpful, optimizing individual benefits.
ASOs are versatile in their usage, as they can be employed to (1) downregulate transcripts in the case of toxic gain-of-function (GoF) and dominant-negative variants, (2) restore the reading frame in the case of truncating variants leading to a loss-of-function (LoF) effect, (3) correct aberrant splicing, and (4) increase protein expression of the wild-type (WT) allele in disorders associated with haploinsufficiency (HI; see Data S1).8,9,10 Hence, ASOs can be used to target specific genetic variants present in groups of individuals, with group sizes being as small as n = 1. However, not all genetic variants can be targeted with ASOs; even the ones that can be targeted can be distinguished into more eligible (stronger) and less eligible (weaker) candidates. Thus, it is important to systematically assess every pathogenic DNA variant for its eligibility for ASO treatment to identify the individuals most likely to benefit from such therapies.
Since 2018, multiple groups and organizations have developed customized ASO treatments for individuals targeted to their specific variant, a single-nucleotide polymorphism, or the disease gene in general.11,12,13 These developments have given hope that individualized, disease-modifying therapies might be a realistic option in the near future for others in the rare-disease community. As of January 2025, to our knowledge, 27 individuals have received individualized ASO therapies, and more are under development .
The N=1 Collaborative (N1C) (https://www.n1collaborative.org/) is a global initiative to develop best practices for ultra-rare “n = 1/few” therapy development and promote safe and equitable access for individuals with rare diseases. The N1C patient identification working group (PIWG) is one of several workgroups organized by the N1C. The PIWG focuses on three key areas: (1) identifying suitable genetic variants for ASO development, (2) determining diseases that are prime candidates for genetic therapy, and (3) identifying individuals who are suitable for individualized genetic therapy development.
Individualized ASO therapy development can be split into three distinct parts (see graphical abstract), with the assessment of the individual at the beginning of the development process. This assessment is based upon three main pillars: (1) assessment of the genetic variant for molecular eligibility, (2) assessment of the disease, and (3) assessment of the individual (disease stage, symptoms, and goals). We have discussed the different aspects of this evaluation process extensively elsewhere.8,14,15,16,17,18
To aid with the prioritization of individuals for ASO developments, the PIWG has developed criteria and established a consensus on assessing diagnostic DNA variants for amenability to ASO therapies. The guidelines are intended for clinical geneticists and clinicians working with rare-disease individuals, diagnostic laboratories, researchers, and research institutes working on rare genetic disorders and aim to help them identify and prioritize amenable disease-causing variants so they can assess individuals for further ASO development.14
Here, we describe the development of the consensus guidelines—named the N1C VARIANT (variant assessments toward eligibility for antisense oligonucleotide treatment) guidelines—present the first version of the guidelines, and provide training materials such as example assessments and training videos. We further introduce the “N1C variant eligibility calculator,” which aids with variant evaluations.
